The T cell receptor (TCR)-CD3 complex is composed of a diverse TCR heterodimer non-covalently associated with the invariant CD3 dimers CD3, CD3 , and CD3 . The TCR mediates peptide-MHC (pMHC) recognition, while the associated CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T cell signaling pathways, the mechanisms whereby TCR engagement by pMHC initiates signaling remain a mystery. A key to solving this problem is establishing the spatial organization of the TCR-CD3 complex. Although the extracellular regions of TCR and CD3 are known to interact, all previous attempts to study the TCR-CD3 receptor by X-ray crystallography have been frustrated by the very low affinity of TCR-CD3 interactions in solution, which precludes the formation of stable assemblies for structural analysis. To overcome this obstacle, this project employs a novel strategy combining NMR spectroscopy to map docking sites in solution with in vitro directed evolution by yeast surface display (YSD) to stabilize TCR-CD3 complexes for crystallization. Our objectives are: 1. Determination of the NMR solution structure for the wild-type TCR-CD3 complex. We will employ chemical shift perturbation and paramagnetic relaxation enhancement (PRE) to determine binding epitopes between CD3 and TCR. These data will be used to determine a structure for the TCR-CD3 complex in solution and to guide the design of YSD experiments. Preliminary NMR spectra support the feasibility of defining the wild-type TCR-CD3 interface in solution. 2. Affinity maturation of binding interactions between TCR and CD3. Large mutant libraries of TCR (107-108 independent clones) will be displayed on yeast and sorted by flow cytometry with CD3 or CD3 tetramers to isolate high-affinity TCR variants. Conversely, mutant libraries of CD3 and CD3 will be affinity-selected with TCR tetramers. As a demonstration of the power of this approach, we recently employed YSD to stabilize the extremely weak interaction between human CD4 and MHC class II (KD > 400 M), which enabled us to determine the first crystal structure of a TCR- pMHC-CD4 ternary complex. 3. Assembly and structural analysis of affinity-matured TCR-CD3 complexes. We will pursue crystallization of high-affinity TCR-CD3 complexes. Both binary and ternary complexes will be targeted: TCR-CD3, TCR-CD3 and TCR-CD3 -CD3 . If crystals cannot be obtained, we will use NMR to determine the structure of the affinity-matured TCR-CD3 complex. Structural information on these complexes, determined either by X-ray or NMR, will define the overall spatial organization of the multisubunit TCR-CD3 receptor.